Compositions comprising bacterial strains

ABSTRACT

The invention provides compositions comprising bacterial strains for treating and preventing cancer.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.16/251,462, filed Jan. 18, 2019, which is a continuation of U.S.application Ser. No. 15/803,721, filed on Nov. 3, 2017, which is acontinuation of U.S. application Ser. No. 15/357,850, filed on Nov. 21,2016, which claims priority to GB Application No. 1604924.9, filed onMar. 23, 2016, and to GB Application No. 1520502.4, filed on Nov. 20,2015, all of which are herein incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 18, 2016, isnamed 49455-709-201_P067592XX_sequence_listing.txt and is 3,999,976bytes in size.

TECHNICAL FIELD

This invention is in the field of compositions comprising bacterialstrains isolated from the mammalian digestive tract and the use of suchcompositions in the treatment of disease.

BACKGROUND TO THE INVENTION

The human intestine is thought to be sterile in utero, but it is exposedto a large variety of maternal and environmental microbes immediatelyafter birth. Thereafter, a dynamic period of microbial colonization andsuccession occurs, which is influenced by factors such as delivery mode,environment, diet and host genotype, all of which impact upon thecomposition of the gut microbiota, particularly during early life.Subsequently, the microbiota stabilizes and becomes adult-like [1]. Thehuman gut microbiota contains more than 500-1000 different phylotypesbelonging essentially to two major bacterial divisions, theBacteroidetes and the Firmicutes [2]. The successful symbioticrelationships arising from bacterial colonization of the human gut haveyielded a wide variety of metabolic, structural, protective and otherbeneficial functions. The enhanced metabolic activities of the colonizedgut ensure that otherwise indigestible dietary components are degradedwith release of by-products providing an important nutrient source forthe host. Similarly, the immunological importance of the gut microbiotais well-recognized and is exemplified in germfree animals which have animpaired immune system that is functionally reconstituted following theintroduction of commensal bacteria [3-5].

Dramatic changes in microbiota composition have been documented ingastrointestinal disorders such as inflammatory bowel disease (IBD). Forexample, the levels of Clostridium cluster XIVa bacteria are reduced insubjects with IBD whilst numbers of E. coli are increased, suggesting ashift in the balance of symbionts and pathobionts within the gut [6-9].Interestingly, this microbial dysbiosis is also associated withimbalances in T effector cell populations.

In recognition of the potential positive effect that certain bacterialstrains may have on the animal gut, various strains have been proposedfor use in the treatment of various diseases (see, for example,[10-13]). Also, certain strains, including mostly Lactobacillus andBifidobacterium strains, have been proposed for use in treating variousinflammatory and autoimmune diseases that are not directly linked to theintestines (see [14] and [15] for reviews). However, the relationshipbetween different diseases and different bacterial strains, and theprecise effects of particular bacterial strains on the gut and at asystemic level and on any particular types of diseases, are poorlycharacterized. For example, certain Enterococcus species have beenimplicated in causing cancer [16].

There is a requirement in the art for new methods of treating diseases.There is also a requirement for the potential effects of gut bacteria tobe characterized so that new therapies using gut bacteria can bedeveloped.

SUMMARY OF THE INVENTION

The inventors have developed new therapies for treating and preventingdiseases. In particular, the inventors have developed new therapies fortreating and preventing cancer. In particular, the inventors haveidentified that bacterial strains of the species Enterococcus gallinarumcan be effective for treating and preventing cancer. As described in theexamples, oral administration of compositions comprising Enterococcusgallinarum may reduce tumor size in mouse models of cancer.

In preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the species Enterococcus gallinarum,for use in a method of treating or preventing cancer, such as breast,lung or liver cancer. The inventors have identified that treatment withcompositions comprising a bacterial strain of the species Enterococcusgallinarum can reduce tumor growth in mouse models of breast, lung andliver cancer. In certain embodiments, the composition is for use in amethod of reducing tumor size or preventing tumor growth in thetreatment of cancer. Compositions using Enterococcus gallinarum may beparticularly effective for reducing tumor size or preventing tumorgrowth in the treatment of cancer.

In preferred embodiments of the invention, the bacterial strain in thecomposition is of Enterococcus gallinarum. Closely related strains mayalso be used, such as bacterial strains that have a 16s rRNA sequencethat is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical tothe 16s rRNA sequence of a bacterial strain of Enterococcus gallinarum.Preferably, the bacterial strain has a 16s rRNA sequence that is atleast 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1or 2. Preferably, the sequence identity is to SEQ ID NO:2. Preferably,the bacterial strain for use in the invention has the 16s rRNA sequencerepresented by SEQ ID NO:2.

Accordingly, the invention also provides a composition comprising abacterial strain that has a 16s rRNA sequence that is at least 95%identical to the 16s rRNA sequence of a bacterial strain of Enterococcusgallinarum for use in a method of treating or preventing cancer. Inparticular, the invention provides a composition comprising a bacterialstrain that has a 16s rRNA sequence that is at least 95% identical toSEQ ID NO: 2 for use in a method of treating or preventing cancer. Insome embodiments, the bacterial strain in the composition is not ofEnterococcus gallinarum. In some embodiments, the bacterial strain inthe composition is not of Enterococcus gallinarum, but is a closelyrelated strain.

In certain embodiments, the composition of the invention is for oraladministration. Oral administration of the strains of the invention canbe effective for treating cancer. Also, oral administration isconvenient for patients and practitioners and allows delivery to and/orpartial or total colonization of the intestine.

In certain embodiments, the composition of the invention comprises oneor more pharmaceutically acceptable excipients or carriers.

In certain embodiments, the composition of the invention comprises abacterial strain that has been lyophilized. Lyophilization is aneffective and convenient technique for preparing stable compositionsthat allow delivery of bacteria.

In certain embodiments, the composition comprises a lyoprotectant whichis a pharmaceutically acceptable excipient, diluent, or carrier.

In certain embodiments, the composition comprises a bacterial strainthat has been lyophilized; and further comprises a pharmaceuticallyacceptable excipient, diluent, or carrier.

In certain embodiments, the composition is a lyophilized composition. Insome cases, the lyophilized composition may be reconstituted prior toadministration to a subject. In some cases, the reconstitution is with adiluent described herein. In some cases, the diluent may be sterilewater, sodium chloride solution, or dextrose solution.

In certain embodiments, the invention provides a food product comprisingthe composition as described above.

In certain embodiments, the invention provides a vaccine compositioncomprising the composition as described above.

Additionally, the invention provides a method of treating or preventingcancer, comprising administering a composition comprising a bacterialstrain of the species Enterococcus gallinarum.

In developing the above invention, the inventors have identified andcharacterized a bacterial strain that is particularly useful fortherapy. The Enterococcus gallinarum strain of the invention is shown tobe effective for treating cancer. Therefore, in another aspect, theinvention provides a cell of the Enterococcus gallinarum straindeposited under accession number NCIMB 42488, or a derivative thereof.The invention also provides compositions comprising such cells, orbiologically pure cultures of such cells. The invention also provides acell of the Enterococcus gallinarum strain deposited under accessionnumber NCIMB 42488, or a derivative thereof, for use in therapy, inparticular for cancer. Similarly, the invention provides a cell of abacterial strain that has a 16s rRNA sequence that is at least 95%identical to SEQ ID NO: 2, or a derivative thereof. The invention alsoprovides compositions comprising such cells, or biologically purecultures of such cells. The invention also provides a cell of abacterial strain that has a 16s rRNA sequence that is at least 95%identical to SEQ ID NO:2, or a derivative thereof, for use in therapy,in particular for treating or preventing cancer.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety forall purposes, to the same extent as if each individual publication,patent, or patent application was specifically and individuallyindicated to be incorporated by reference.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Mouse model of breast cancer—tumor volume.

FIG. 2: Mouse model of lung cancer—tumor volume.

FIG. 3: Mouse model of liver cancer—liver weight.

FIG. 4A: Cytokine levels (pg/ml) in immature dendritic cells (Nobacteria).

FIG. 4B: Cytokine levels (pg/ml) in immature dendritic cells after theaddition of LPS.

FIG. 4C: Cytokine levels (pg/ml) in immature dendritic cells after theaddition of MRX518.

FIG. 4D: Cytokine levels (pg/ml) in immature dendritic cells after theaddition of MRX518 and LPS.

FIG. 5A: Cytokine levels in THP-1 cells (No bacteria).

FIG. 5B: Cytokine levels in THP-1 cells after addition of bacterialsediment.

FIG. 5C: Cytokine levels in THP-1 cells after the addition of MRX518alone or in combination with LPS.

DETAILED DESCRIPTION

Bacterial Strains

The compositions of the invention comprise a bacterial strain of thespecies Enterococcus gallinarum. The examples demonstrate that bacteriaof this species are useful for treating or preventing cancer.

The invention also provides compositions comprising a bacterial strainthat has a 16s rRNA sequence that is at least 95% identical to the 16srRNA sequence of a bacterial strain of Enterococcus gallinarum for usein therapy, for example, for use in a method of treating or preventingcancer. In particular, the invention also provides compositionscomprising a bacterial strain that has a 16s rRNA sequence that is atleast 95% identical to SEQ ID NO: 2 for use in therapy, for example, foruse in a method of treating or preventing cancer. In some embodiments,the bacterial strain in the composition is not of Enterococcusgallinarum, but is a closely related strain.

The invention provides an Enterococcus gallinarum for use in therapy,for example, for use in treating or preventing cancer. Similarly, theinvention provides a composition comprising a bacterial strain of thespecies Enterococcus gallinarum, for use in therapy, for example, foruse in treating or preventing cancer. In certain embodiments, thecompositions of the invention comprise a bacterial strain that has a 16srRNA sequence that is at least 95% identical to SEQ ID NO: 2, forexample which is a Enterococcus gallinarum, and do not contain any otherbacterial genus. In certain embodiments, the compositions of theinvention comprise a single strain of a bacterial strain that has a 16srRNA sequence that is at least 95% identical to SEQ ID NO: 2, forexample, which is an Enterococcus gallinarum, and do not contain anyother bacterial strain or species.

Enterococcus gallinarum forms coccoid cells, mostly in pairs or shortchains. It is nonmotile and colonies on blood agar or nutrient agar arecircular and smooth. Enterococcus gallinarum reacts with Lancefieldgroup D antisera. The type strain of Enterococcus gallinarum isF87/276=PB21=ATCC 49573=CCUG 18658=CIP 103013=JCM 8728=LMG 13129=NBRC100675=NCIMB 702313 (formerly NCDO 2313)=NCTC 12359 [17]. The GenBankaccession number for a 16S rRNA gene sequence of Enterococcus gallinarumis AF039900 (disclosed herein as SEQ ID NO:1). An exemplary Enterococcusgallinarum strain is described in [17].

All microorganism deposits were made under the terms of the BudapestTreaty. Maintenance of a viable culture is assured for 30 years from thedate of deposit. All restrictions on the availability to the public ofthe deposited microorganisms will be irrevocably removed upon thegranting of a patent for this application. The Enterococcus gallinarumbacterium deposited under accession number NCIMB 42488 was tested in theExamples and is also referred to herein as strain MRX518. References toMRX518 and MRx0518 are used interchangeably. A 16S rRNA sequence for theMRX518 strain that was tested is provided in SEQ ID NO:2. Strain MRX518was deposited with the international depositary authority NCIMB, Ltd.(Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma ResearchLtd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland)on 16 Nov. 2015 as “Enterococcus sp” and was assigned accession numberNCIMB 42488.

The genome of strain MRX518 comprises a chromosome and plasmid. Achromosome sequence for strain MRX518 is provided in SEQ ID NO:3. Aplasmid sequence for strain MRX518 is provided in SEQ ID NO:4. Thesesequences were generated using the PacBio RS II platform.

Bacterial strains closely related to the strain tested in the examplesare also expected to be effective for treating or preventing cancer. Incertain embodiments, the bacterial strain for use in the invention has a16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or99.9% identical to the 16s rRNA sequence of a bacterial strain ofEnterococcus gallinarum. Preferably, the bacterial strain for use in theinvention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to SEQ ID NO:1 or 2. Preferably, thesequence identity is to SEQ ID NO:2. Preferably, the bacterial strainfor use in the invention has the 16s rRNA sequence represented by SEQ IDNO:2.

Bacterial strains that are biotypes of the bacterium deposited underaccession number 42488 are also expected to be effective for treating orpreventing cancer. A biotype is a closely related strain that has thesame or very similar physiological and biochemical characteristics.

Strains that are biotypes of the bacterium deposited under accessionnumber NCIMB 42488 and that are suitable for use in the invention may beidentified by sequencing other nucleotide sequences for the bacteriumdeposited under accession number NCIMB 42488. For example, substantiallythe whole genome may be sequenced and a biotype strain for use in theinvention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%sequence identity across at least 80% of its whole genome (e.g. acrossat least 85%, 90%, 95% or 99%, or across its whole genome). For example,in some embodiments, a biotype strain has at least 98% sequence identityacross at least 98% of its genome or at least 99% sequence identityacross 99% of its genome. Other suitable sequences for use inidentifying biotype strains may include hsp60 or repetitive sequencessuch as BOX, ERIC, (GTG)₅, or REP or [18]. Biotype strains may havesequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequenceidentity to the corresponding sequence of the bacterium deposited underaccession number NCIMB 42488. In some embodiments, a biotype strain hasa sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%sequence identity to the corresponding sequence of strain MRX518deposited as NCIMB 42488 and comprises a 16S rRNA sequence that is atleast 99% identical (e.g. at least 99.5% or at least 99.9% identical) toSEQ ID NO:2. In some embodiments, a biotype strain has a sequence withat least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity tothe corresponding sequence of strain MRX518 deposited as NCIMB 42488 andhas the 16S rRNA sequence of SEQ ID NO:2.

In certain embodiments, the bacterial strain for use in the inventionhas a chromosome with sequence identity to SEQ ID NO:3. In preferredembodiments, the bacterial strain for use in the invention has achromosome with at least 90% sequence identity (e.g. at least 92%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:3 acrossat least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%,99% or 100%) of SEQ ID NO:3. For example, the bacterial strain for usein the invention may have a chromosome with at least 90% sequenceidentity to SEQ ID NO:3 across 70% of SEQ ID NO:3, or at least 90%sequence identity to SEQ ID NO:3 across 80% of SEQ ID NO:3, or at least90% sequence identity to SEQ ID NO:3 across 90% of SEQ ID NO:3, or atleast 90% sequence identity to SEQ ID NO:3 across 100% of SEQ ID NO:3,or at least 95% sequence identity to SEQ ID NO:3 across 70% of SEQ IDNO:3, or at least 95% sequence identity to SEQ ID NO:3 across 80% of SEQID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 90% ofSEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across100% of SEQ ID NO:3, or at least 98% sequence identity to SEQ ID NO:3across 70% of SEQ ID NO:3, or at least 98% sequence identity to SEQ IDNO:3 across 80% of SEQ ID NO:3, or at least 98% sequence identity to SEQID NO:3 across 90% of SEQ ID NO:3, or at least 98% identity to SEQ IDNO:3 across 95% of SEQ ID NO:3, or at least 98% sequence identity to SEQID NO:3 across 100% of SEQ ID NO:3, or at least 99.5% sequence identityto SEQ ID NO:3 across 90% of SEQ ID NO:3, or at least 99.5% identity toSEQ ID NO:3 across 95% of SEQ ID NO:3, or at least 99.5% identity to SEQID NO:3 across 98% of SEQ ID NO:3, or at least 99.5% sequence identityto SEQ ID NO:3 across 100% of SEQ ID NO:3.

In certain embodiments, the bacterial strain for use in the inventionhas a plasmid with sequence identity to SEQ ID NO:4. In preferredembodiments, the bacterial strain for use in the invention has a plasmidwith at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:4 across at least60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or100%) of SEQ ID NO:4. For example, the bacterial strain for use in theinvention may have a plasmid with at least 90% sequence identity to SEQID NO:4 across 70% of SEQ ID NO:4, or at least 90% sequence identity toSEQ ID NO:4 across 80% of SEQ ID NO:4, or at least 90% sequence identityto SEQ ID NO:4 across 90% of SEQ ID NO:4, or at least 90% sequenceidentity to SEQ ID NO:4 across 100% of SEQ ID NO:4, or at least 95%sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least95% sequence identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or atleast 95% sequence identity to SEQ ID NO:4 across 90% of SEQ ID NO:4, orat least 95% sequence identity to SEQ ID NO:4 across 100% of SEQ IDNO:4, or at least 98% sequence identity to SEQ ID NO:4 across 70% of SEQID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 80% ofSEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 90%of SEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across100% of SEQ ID NO:4.

In certain embodiments, the bacterial strain for use in the inventionhas a chromosome with sequence identity to SEQ ID NO:3 and a plasmidwith sequence identity to SEQ ID NO:4.

In certain embodiments, the bacterial strain for use in the inventionhas a chromosome with sequence identity to SEQ ID NO:3, for example asdescribed above, and a 16S rRNA sequence with sequence identity to anyof SEQ ID NO:1 or 2, for example as described above, preferably with a16s rRNA sequence that is at least 99% identical to SEQ ID NO: 2, morepreferably which comprises the 16S rRNA sequence of SEQ ID NO:2, andoptionally comprises a plasmid with sequence identity to SEQ ID NO:4, asdescribed above.

In certain embodiments, the bacterial strain for use in the inventionhas a chromosome with sequence identity to SEQ ID NO:3, for example asdescribed above, and optionally comprises a plasmid with sequenceidentity to SEQ ID NO:4, as described above, and is effective fortreating or preventing cancer.

In certain embodiments, the bacterial strain for use in the inventionhas a chromosome with sequence identity to SEQ ID NO:3, for example asdescribed above, and a 16S rRNA sequence with sequence identity to anyof SEQ ID NOs: 1 or 2, for example as described above, and optionallycomprises a plasmid with sequence identity to SEQ ID NO:4, as describedabove, and is effective for treating or preventing cancer.

In certain embodiments, the bacterial strain for use in the inventionhas a 16s rRNA sequence that is at least 99%, 99.5% or 99.9% identicalto the 16s rRNA sequence represented by SEQ ID NO: 2 (for example, whichcomprises the 16S rRNA sequence of SEQ ID NO:2) and a chromosome with atleast 95% sequence identity to SEQ ID NO:3 across at least 90% of SEQ IDNO:3, and optionally comprises a plasmid with sequence identity to SEQID NO:4, as described above, and which is effective for treating orpreventing cancer.

In certain embodiments, the bacterial strain for use in the inventionhas a 16s rRNA sequence that is at least 99%, 99.5% or 99.9% identicalto the 16s rRNA sequence represented by SEQ ID NO: 2 (for example, whichcomprises the 16S rRNA sequence of SEQ ID NO:2) and a chromosome with atleast 98% sequence identity (e.g. at least 99% or at least 99.5%sequence identity) to SEQ ID NO:3 across at least 98% (e.g. across atleast 99% or at least 99.5%) of SEQ ID NO:3, and optionally comprises aplasmid with sequence identity to SEQ ID NO:4, as described above, andwhich is effective for treating or preventing cancer.

In certain embodiments, the bacterial strain for use in the invention isa Enterococcus gallinarum and has a 16s rRNA sequence that is at least99%, 99.5% or 99.9% identical to the 16s rRNA sequence represented bySEQ ID NO: 2 (for example, which comprises the 16S rRNA sequence of SEQID NO:2) and a chromosome with at least 98% sequence identity (e.g. atleast 99% or at least 99.5% sequence identity) to SEQ ID NO:3 across atleast 98% (e.g. across at least 99% or at least 99.5%) of SEQ ID NO:3,and optionally comprises a plasmid with sequence identity to SEQ IDNO:4, as described above, and which is effective for treating orpreventing cancer.

Alternatively, strains that are biotypes of the bacterium depositedunder accession number NCIMB 42488 and that are suitable for use in theinvention may be identified by using the accession number NCIMB 42488deposit and restriction fragment analysis and/or PCR analysis, forexample by using fluorescent amplified fragment length polymorphism(FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or proteinprofiling, or partial 16S or 23s rDNA sequencing. In preferredembodiments, such techniques may be used to identify other Enterococcusgallinarum strains.

In certain embodiments, strains that are biotypes of the bacteriumdeposited under accession number NCIMB 42488 and that are suitable foruse in the invention are strains that provide the same pattern as thebacterium deposited under accession number NCIMB 42488 when analyzed byamplified ribosomal DNA restriction analysis (ARDRA), for example whenusing Sau3AI restriction enzyme (for exemplary methods and guidance see,for example, [19]). Alternatively, biotype strains are identified asstrains that have the same carbohydrate fermentation patterns as thebacterium deposited under accession number NCIMB 42488. In someembodiments, the carbohydrate fermentation pattern is determined usingthe API 50 CHL panel (bioMérieux). In some embodiments, the bacterialstrain used in the invention is:

-   -   positive for fermentation of at least one of (e.g. at least 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or all of):        L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose,        D-fructose, D-mannose, N-acetylglucosamine, amygdalin, arbutin,        salicin, D-cellobiose, D-maltose, sucrose, D-trehalose,        gentiobiose, D-tagatose and potassium gluconate; and/or    -   intermediate for fermentation of at least one of (e.g. at least        2, 3, 4 or all of): D-mannitol, Methyl-αD-glycopyranoside,        D-lactose, starch, and L-fucose;        preferably as determined by API 50 CHL analysis (preferably        using the API 50 CHL panel from bioMérieux).

Other Enterococcus gallinarum strains that are useful in thecompositions and methods of the invention, such as biotypes of thebacterium deposited under accession number NCIMB 42488, may beidentified using any appropriate method or strategy, including theassays described in the examples. For instance, strains for use in theinvention may be identified by culturing in anaerobic YCFA and/oradministering the bacteria to the type II collagen-induced arthritismouse model and then assessing cytokine levels. In particular, bacterialstrains that have similar growth patterns, metabolic type and/or surfaceantigens to the bacterium deposited under accession number NCIMB 42488may be useful in the invention. A useful strain will have comparableimmune modulatory activity to the NCIMB 42488 strain. In particular, abiotype strain will elicit comparable effects on the cancer diseasemodels to the effects shown in the Examples, which may be identified byusing the culturing and administration protocols described in theExamples.

In some embodiments, the bacterial strain used in the invention is:

-   -   Positive for at least one of (e.g. at least 2, 3, 4, 5, 6, 7 or        all of): mannose fermentation, glutamic acid decarboxylase,        arginine arylamidase, phenylalanine arylamidase, pyroglutamic        acid arylamidase, tyrosine arylamidase, histidine arylamidase        and serine arylamidase; and/or    -   Intermediate for at least one of (e.g. at least 2 or all of):        β-galactosidase-6-phosphate, β-glucosidase and        N-acetyl-β-glucosaminidase; and/or    -   Negative for at least one of (e.g. at least 2, 3, 4, 5, 6 or all        of): Raffinose fermentation, Proline arylamidase, Leucyl glycine        arylamidase, Leucine arylamidase, Alanine arylamidase, Glycine        arylamidase and Glutamyl glutamic acid arylamidase,        preferably as determined by an assay of carbohydrate, amino acid        and nitrate metabolism, and optionally an assay of alkaline        phosphatase activity, more preferably as determined by Rapid ID        32A analysis (preferably using the Rapid ID 32A system from        bioMérieux).

In some embodiments, the bacterial strain used in the invention is:

-   -   Negative for at least one of (e.g. at least 2, 3, or all 4 of)        glycine arylamidase, raffinose fermentation, proline        arylamidase, and leucine arylamidase, for example, as determined        by an assay of carbohydrate, amino acid and nitrate metabolism,        preferably as determined by Rapid ID 32A analysis (preferably        using the Rapid ID 32A system from bioMérieux); and/or    -   Intermediate positive for fermentation of L-fucose, preferably        as determined by API 50 CHL analysis (preferably using the API        50 CHL panel from bioMérieux).

In some embodiments, the bacterial strain used in the invention is anextracellular ATP producer, for example one which produces 6-6.7 ng/μl(for example, 6.1-6.6 ng/μl or 6.2-6.5 ng/μl or 6.33±0.10 ng/μl) of ATPas measured using the ATP Assay Kit (Sigma-Aldrich, MAK190). Bacterialextracellular ATP can have pleiotropic effects including activation of Tcell-receptor mediated signalling (Schenk et al., 2011), promotion ofintestinal Th17 cell differentiation (Atarashi et al., 2008) andinduction of secretion of the pro-inflammatory mediator IL-1β byactivating the NLRP3 inflammasome (Karmarkar et al., 2016). Accordingly,a bacterial strain which is an extracellular ATP producer is useful fortreating or preventing cancer.

In some embodiments, the bacterial strain for use in the inventioncomprises one or more of the following three genes: Mobile elementprotein; Xylose ABC transporter, permease component; and FIG00632333:hypothetical protein. For example, in certain embodiments, the bacterialstrain for use in the invention comprises genes encoding Mobile elementprotein and Xylose ABC transporter, permease component; Mobile elementprotein and FIG00632333: hypothetical protein; Xylose ABC transporter,permease component and FIG00632333: hypothetical protein; or Mobileelement protein, Xylose ABC transporter, permease component, andFIG00632333: hypothetical protein.

A particularly preferred strain of the invention is the Enterococcusgallinarum strain deposited under accession number NCIMB 42488. This isthe exemplary MRX518 strain tested in the examples and shown to beeffective for treating disease. Therefore, the invention provides acell, such as an isolated cell, of the Enterococcus gallinarum straindeposited under accession number NCIMB 42488, or a derivative thereof.The invention also provides a composition comprising a cell of theEnterococcus gallinarum strain deposited under accession number NCIMB42488, or a derivative thereof. The invention also provides abiologically pure culture of the Enterococcus gallinarum straindeposited under accession number NCIMB 42488. The invention alsoprovides a cell of the Enterococcus gallinarum strain deposited underaccession number NCIMB 42488, or a derivative thereof, for use intherapy, in particular for the diseases described herein. A derivativeof the strain deposited under accession number NCIMB 42488 may be adaughter strain (progeny) or a strain cultured (subcloned) from theoriginal.

A derivative of a strain of the invention may be modified, for exampleat the genetic level, without ablating the biological activity. Inparticular, a derivative strain of the invention is therapeuticallyactive. A derivative strain will have comparable immune modulatoryactivity to the original NCIMB 42488 strain. In particular, a derivativestrain will elicit comparable effects on the cancer disease models tothe effects shown in the Examples, which may be identified by using theculturing and administration protocols described in the Examples. Aderivative of the NCIMB 42488 strain will generally be a biotype of theNCIMB 42488 strain.

References to cells of the Enterococcus gallinarum strain depositedunder accession number NCIMB 42488 encompass any cells that have thesame safety and therapeutic efficacy characteristics as the strainsdeposited under accession number NCIMB 42488, and such cells areencompassed by the invention. Thus, in some embodiments, reference tocells of the Enterococcus gallinarum strain deposited under accessionnumber NCIMB 42488 refers only to the MRX518 strain deposited underNCIMB 42488 and does not refer to a bacterial strain that was notdeposited under NCIMB 42488. In some embodiments, reference to cells ofthe Enterococcus gallinarum strain deposited under accession numberNCIMB 42488 refers to cells that have the same safety and therapeuticefficacy characteristics as the strains deposited under accession numberNCIMB 42488, but which are not the strain deposited under NCIMB 42488.

In preferred embodiments, the bacterial strains in the compositions ofthe invention are viable and capable of partially or totally colonizingthe intestine.

Treating Cancer

In preferred embodiments, the compositions of the invention are for usein treating or preventing cancer. The examples demonstrate thatadministration of the compositions of the invention can lead to areduction in tumor growth in a number of tumor models.

In certain embodiments, treatment with the compositions of the inventionresults in a reduction in tumor size or a reduction in tumor growth. Incertain embodiments, the compositions of the invention are for use inreducing tumor size or reducing tumor growth. The compositions of theinvention may be effective for reducing tumor size or growth. In certainembodiments, the compositions of the invention are for use in subjectswith solid tumors. In certain embodiments, the compositions of theinvention are for use in reducing or preventing angiogenesis in thetreatment of cancer. The compositions of the invention may have aneffect on the immune or inflammatory systems, which have central rolesin angiogenesis. In certain embodiments, the compositions of theinvention are for use in preventing metastasis.

In certain embodiments, the compositions of the invention are for use intreating or preventing breast cancer. The examples demonstrate that thecompositions of the invention may be effective for treating breastcancer. In certain embodiments, the compositions of the invention arefor use in reducing tumor size, reducing tumor growth, or reducingangiogenesis in the treatment of breast cancer. In preferred embodimentsthe cancer is mammary carcinoma. In preferred embodiments the cancer isstage IV breast cancer.

In certain embodiments, the compositions of the invention are for use intreating or preventing lung cancer. The examples demonstrate that thecompositions of the invention may be effective for treating lung cancer.In certain embodiments, the compositions of the invention are for use inreducing tumor size, reducing tumor growth, or reducing angiogenesis inthe treatment of lung cancer. In preferred embodiments the cancer islung carcinoma.

In certain embodiments, the compositions of the invention are for use intreating or preventing liver cancer. The examples demonstrate that thecompositions of the invention may be effective for treating livercancer. In certain embodiments, the compositions of the invention arefor use in reducing tumor size, reducing tumor growth, or reducingangiogenesis in the treatment of liver cancer. In preferred embodimentsthe cancer is hepatoma (hepatocellular carcinoma).

In certain embodiments, the compositions of the invention are for use intreating or preventing colon cancer. The examples demonstrate that thecompositions of the invention have an effect on colon cancer cells andmay be effective for treating colon cancer. In certain embodiments, thecompositions of the invention are for use in reducing tumor size,reducing tumor growth, or reducing angiogenesis in the treatment ofcolon cancer. In preferred embodiments the cancer is colorectaladenocarcinoma.

In some embodiments, the cancer is of the intestine. In someembodiments, the cancer is of a part of the body which is not theintestine. In some embodiments, the cancer is not cancer of theintestine. In some embodiments, the cancer is not colorectal cancer. Insome embodiments, the cancer is not cancer of the small intestine. Insome embodiments, the treating or preventing occurs at a site other thanat the intestine. In some embodiments, the treating or preventing occursat the intestine and also at a site other than at the intestine.

In certain embodiments, the compositions of the invention are for use intreating or preventing carcinoma. The examples demonstrate that thecompositions of the invention may be effective for treating numeroustypes of carcinoma. In certain embodiments, the compositions of theinvention are for use in treating or preventing non-immunogenic cancer.The examples demonstrate that the compositions of the invention may beeffective for treating non-immunogenic cancers.

The therapeutic effects of the compositions of the invention on cancermay be mediated by a pro-inflammatory mechanism. Examples 2, 4 and 5demonstrate that the expression of a number of pro-inflammatorycytokines may be increased following administration of MRX518.Inflammation can have a cancer-suppressive effect [20] andpro-inflammatory cytokines such as TNFα are being investigated as cancertherapies [21]. The up-regulation of genes such as TNF shown in theexamples may indicate that the compositions of the invention may beuseful for treating cancer via a similar mechanism. The up-regulation ofCXCR3 ligands (CXCL9, CXCL10) and IFNγ-inducible genes (IL-32) mayindicate that the compositions of the invention elicit an IFNγ-typeresponse. IFNγ is a potent macrophage-activating factor that canstimulate tumirocidal activity [22], and CXCL9 and CXCL10, for example,also have anti-cancer effects [23-25]. Therefore, in certainembodiments, the compositions of the invention are for use in promotinginflammation in the treatment of cancer. In preferred embodiments, thecompositions of the invention are for use in promoting Th1 inflammationin the treatment of cancer. Th1 cells produce IFNγ and have potentanti-cancer effects [20]. In certain embodiments, the compositions ofthe invention are for use in treating an early-stage cancer, such as acancer that has not metastasized, or a stage 0 or stage 1 cancer.Promoting inflammation may be more effective against early-stage cancers[20]. In certain embodiments, the compositions of the invention are foruse in promoting inflammation to enhance the effect of a secondanti-cancer agent. In certain embodiments, the treatment or preventionof cancer comprises increasing the level of expression of one or morecytokines. For example, in certain embodiments, the treatment orprevention of cancer comprises increasing the level of expression of oneor more of IL-1β, IL-6 and TNF-α, for example, IL-1β and IL-6, IL-1β andTNF-α, IL-6 and TNF-α or all three of IL-1β, IL-6 and TNF-α. Increasesin levels of expression of any of IL-1β, IL-6 and TNF-α are known to beindicative of efficacy in treatment of cancer.

Examples 4 and 5 demonstrate that when a bacterial strain as describedherein is used in combination with lipopolysaccharide (LPS), there is asynergistic increase in IL-1β. LPS is known to elicit a pro-inflammatoryeffect. Thus, in certain embodiments, the treatment or preventioncomprises using a bacterial strain as described herein in combinationwith an agent that upregulates IL-1β. In certain embodiments, thetreatment or prevention comprises using a bacterial strain as describedherein in combination with LPS. Accordingly, a composition of theinvention may additionally comprise an agent that upregulates IL-1β.Accordingly, a composition of the invention may additionally compriseLPS.

In certain embodiments, the compositions of the invention are for use intreating a subject that has previously received chemotherapy. In certainembodiments, the compositions of the invention are for use in treating asubject that has not tolerated a chemotherapy treatment. Thecompositions of the invention may be particularly suitable for suchsubjects. In some instances, a subject is in need thereof.

In certain embodiments, the compositions of the invention are forpreventing relapse. The compositions of the invention may be suitablefor long-term administration. In certain embodiments, the compositionsof the invention are for use in preventing progression of cancer.

In certain embodiments, the compositions of the invention are for use intreating non-small-cell lung carcinoma. In certain embodiments, thecompositions of the invention are for use in treating small-cell lungcarcinoma. In certain embodiments, the compositions of the invention arefor use in treating squamous-cell carcinoma. In certain embodiments, thecompositions of the invention are for use in treating adenocarcinoma. Incertain embodiments, the compositions of the invention are for use intreating glandular tumors, carcinoid tumors, or undifferentiatedcarcinomas.

In certain embodiments, the compositions of the invention are for use intreating hepatoblastoma, cholangiocarcinoma, cholangiocellularcystadenocarcinoma or liver cancer resulting from a viral infection.

In certain embodiments, the compositions of the invention are for use intreating invasive ductal carcinoma, ductal carcinoma in situ or invasivelobular carcinoma.

In further embodiments, the compositions of the invention are for use intreating or preventing acute lymphoblastic leukemia (ALL), acute myeloidleukemia, adrenocortical carcinoma, basal-cell carcinoma, bile ductcancer, bladder cancer, bone tumor, osteosarcoma/malignant fibroushistiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma,cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, breast cancer,bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumor,cervical cancer, chronic lymphocytic leukemia, chronic myelogenousleukemia, chronic myeloproliferative disorders, colon cancer, cutaneousT-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer,Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladdercancer, gastric cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor (GIST), germ cell tumor, glioma,childhood visual pathway and hypothalamic, Hodgkin lymphoma, melanoma,islet cell carcinoma, Kaposi sarcoma, renal cell cancer, laryngealcancer, leukaemias, lymphomas, mesothelioma, neuroblastoma, non-Hodgkinlymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreaticcancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasmacell neoplasia, prostate cancer, renal cell carcinoma, retinoblastoma,sarcoma, testicular cancer, thyroid cancer, or uterine cancer.

The compositions of the invention may be particularly effective whenused in combination with further therapeutic agents. Theimmune-modulatory effects of the compositions of the invention may beeffective when combined with more direct anti-cancer agents. Therefore,in certain embodiments, the invention provides a composition comprisinga bacterial strain of the species Enterococcus gallinarum and ananticancer agent. In preferred embodiments the anticancer agent is animmune checkpoint inhibitor, a targeted antibody immunotherapy, a CAR-Tcell therapy, an oncolytic virus, or a cytostatic drug. In preferredembodiments, the composition comprises an anti-cancer agent selectedfrom the group consisting of: Yervoy (ipilimumab, BMS); Keytruda(pembrolizumab, Merck); Opdivo (nivolumab, BMS); MEDI4736(AZ/MedImmune); MPDL3280A (Roche/Genentech); Tremelimumab(AZ/MedImmune); CT-011 (pidilizumab, CureTech); BMS-986015 (lirilumab,BMS); MEDI0680 (AZ/MedImmune); MSB-0010718C (Merck); PF-05082566(Pfizer); MEDI6469 (AZ/MedImmune); BMS-986016 (BMS); BMS-663513(urelumab, BMS); IMP321 (Prima Biomed); LAG525 (Novartis); ARGX-110(arGEN-X); PF-05082466 (Pfizer); CDX-1127 (varlilumab; CellDexTherapeutics); TRX-518 (GITR Inc.); MK-4166 (Merck); JTX-2011 (JounceTherapeutics); ARGX-115 (arGEN-X); NLG-9189 (indoximod, NewLinkGenetics); INCB024360 (Incyte); IPH2201 (Innate Immotherapeutics/AZ);NLG-919 (NewLink Genetics); anti-VISTA (JnJ); Epacadostat (INCB24360,Incyte); F001287 (Flexus/BMS); CP 870893 (University of Pennsylvania);MGA271 (Macrogenix); Emactuzumab (Roche/Genentech); Galunisertib (EliLilly); Ulocuplumab (BMS); BKT140/BL8040 (Biokine Therapeutics);Bavituximab (Peregrine Pharmaceuticals); CC 90002 (Celgene); 852A(Pfizer); VTX-2337 (VentiRx Pharmaceuticals); IMO-2055 (Hybridon, IderaPharmaceuticals); LY2157299 (Eli Lilly); EW-7197 (Ewha Women'sUniversity, Korea); Vemurafenib (Plexxikon); Dabrafenib (Genentech/GSK);BMS-777607 (BMS); BLZ945 (Memorial Sloan-Kettering Cancer Centre);Unituxin (dinutuximab, United Therapeutics Corporation); Blincyto(blinatumomab, Amgen); Cyramza (ramucirumab, Eli Lilly); Gazyva(obinutuzumab, Roche/Biogen); Kadcyla (ado-trastuzumab emtansine,Roche/Genentech); Perjeta (pertuzumab, Roche/Genentech); Adcetris(brentuximab vedotin, Takeda/Millennium); Arzerra (ofatumumab, GSK);Vectibix (panitumumab, Amgen); Avastin (bevacizumab, Roche/Genentech);Erbitux (cetuximab, BMS/Merck); Bexxar (tositumomab-I131, GSK); Zevalin(ibritumomab tiuxetan, Biogen); Campath (alemtuzumab, Bayer); Mylotarg(gemtuzumab ozogamicin, Pfizer); Herceptin (trastuzumab,Roche/Genentech); Rituxan (rituximab, Genentech/Biogen); volociximab(Abbvie); Enavatuzumab (Abbvie); ABT-414 (Abbvie); Elotuzumab(Abbvie/BMS); ALX-0141 (Ablynx); Ozaralizumab (Ablynx); Actimab-C(Actinium); Actimab-P (Actinium); Milatuzumab-dox (Actinium); Emab-SN-38(Actinium); Naptumonmab estafenatox (Active Biotech); AFM13 (Affimed);AFM11 (Affimed); AGS-16C3F (Agensys); AGS-16M8F (Agensys); AGS-22ME(Agensys); AGS-15ME (Agensys); GS-67E (Agensys); ALXN6000 (samalizumab,Alexion); ALT-836 (Altor Bioscience); ALT-801 (Altor Bioscience);ALT-803 (Altor Bioscience); AMG780 (Amgen); AMG 228 (Amgen); AMG820(Amgen); AMG172 (Amgen); AMG595 (Amgen); AMG110 (Amgen); AMG232(adecatumumab, Amgen); AMG211 (Amgen/MedImmune); BAY20-10112(Amgen/Bayer); Rilotumumab (Amgen); Denosumab (Amgen); AMP-514 (Amgen);MEDI575 (AZ/MedImmune); MEDI3617 (AZ/MedImmune); MEDI6383(AZ/MedImmune); MEDI551 (AZ/MedImmune); Moxetumomab pasudotox(AZ/Medlmmune); MEDI565 (AZ/Medlmmune); MEDI0639 (AZ/Medlmmune);MEDI0680 (AZ/MedImmune); MEDI562 (AZ/MedImmune); AV-380 (AVEO); AV203(AVEO); AV299 (AVEO); BAY79-4620 (Bayer); Anetumab ravtansine (Bayer);vantictumab (Bayer); BAY94-9343 (Bayer); Sibrotuzumab (BoehringerIngleheim); BI-836845 (Boehringer Ingleheim); B-701 (BioClin); BIIB015(Biogen); Obinutuzumab (Biogen/Genentech); BI-505 (Bioinvent); BI-1206(Bioinvent); TB-403 (Bioinvent); BT-062 (Biotest) BIL-010t (Biosceptre);MDX-1203 (BMS); MDX-1204 (BMS); Necitumumab (BMS); CAN-4 (Cantargia AB);CDX-011 (Celldex); CDX1401 (Celldex); CDX301 (Celldex); U3-1565 (DaiichiSankyo); patritumab (Daiichi Sankyo); tigatuzumab (Daiichi Sankyo);nimotuzumab (Daiichi Sankyo); DS-8895 (Daiichi Sankyo); DS-8873 (DaiichiSankyo); DS-5573 (Daiichi Sankyo); MORab-004 (Eisai); MORab-009 (Eisai);MORab-003 (Eisai); MORab-066 (Eisai); LY3012207 (Eli Lilly); LY2875358(Eli Lilly); LY2812176 (Eli Lilly); LY3012217 (Eli Lilly); LY2495655(Eli Lilly); LY3012212 (Eli Lilly); LY3012211 (Eli Lilly); LY3009806(Eli Lilly); cixutumumab (Eli Lilly); Flanvotumab (Eli Lilly); IMC-TR1(Eli Lilly); Ramucirumab (Eli Lilly); Tabalumab (Eli Lilly); Zanolimumab(Emergent Biosolution); FG-3019 (FibroGen); FPA008 (Five PrimeTherapeutics); FP-1039 (Five Prime Therapeutics); FPA144 (Five PrimeTherapeutics); catumaxomab (Fresenius Biotech); IMAB362 (Ganymed);IMAB027 (Ganymed); HuMax-CD74 (Genmab); HuMax-TFADC (Genmab); GS-5745(Gilead); GS-6624 (Gilead); OMP-21M18 (demcizumab, GSK); mapatumumab(GSK); IMGN289 (ImmunoGen); IMGN901 (ImmunoGen); IMGN853 (ImmunoGen);IMGN529 (ImmunoGen); IMMU-130 (Immunomedics); milatuzumab-dox(Immunomedics); IMMU-115 (Immunomedics); IMMU-132 (Immunomedics);IMMU-106 (Immunomedics); IMMU-102 (Immunomedics); Epratuzumab(Immunomedics); Clivatuzumab (Immunomedics); IPH41 (InnateImmunotherapeutics); Daratumumab (Janssen/Genmab); CNTO-95 (Intetumumab,Janssen); CNTO-328 (siltuximab, Janssen); KB004 (KaloBios);mogamulizumab (Kyowa Hakko Kirrin); KW-2871 (ecromeximab, Life Science);Sonepcizumab (Lpath); Margetuximab (Macrogenics); Enoblituzumab(Macrogenics); MGD006 (Macrogenics); MGF007 (Macrogenics); MK-0646(dalotuzumab, Merck); MK-3475 (Merck); Sym004 (Symphogen/Merck Serono);DI17E6 (Merck Serono); MOR208 (Morphosys); MOR202 (Morphosys); Xmab5574(Morphosys); BPC-1C (ensituximab, Precision Biologics); TAS266(Novartis); LFA102 (Novartis); BHQ880 (Novartis/Morphosys); QGE031(Novartis); HCD122 (lucatumumab, Novartis); LJM716 (Novartis); AT355(Novartis); OMP-21M18 (Demcizumab, OncoMed); OMP52M51 (Oncomed/GSK);OMP-59R5 (Oncomed/GSK); vantictumab (Oncomed/Bayer); CMC-544 (inotuzumabozogamicin, Pfizer); PF-03446962 (Pfizer); PF-04856884 (Pfizer);PSMA-ADC (Progenics); REGN1400 (Regeneron); REGN910 (nesvacumab,Regeneron/Sanofi); REGN421 (enoticumab, Regeneron/Sanofi); RG7221,RG7356, RG7155, RG7444, RG7116, RG7458, RG7598, RG7599, RG7600, RG7636,RG7450, RG7593, RG7596, DCDS3410A, RG7414 (parsatuzumab), RG7160(imgatuzumab), RG7159 (obintuzumab), RG7686, RG3638 (onartuzumab),RG7597 (Roche/Genentech); SAR307746 (Sanofi); SAR566658 (Sanofi);SAR650984 (Sanofi); SAR153192 (Sanofi); SAR3419 (Sanofi); SAR256212(Sanofi), SGN-LIVIA (lintuzumab, Seattle Genetics); SGN-CD33A (SeattleGenetics); SGN-75 (vorsetuzumab mafodotin, Seattle Genetics); SGN-19A(Seattle Genetics) SGN-CD70A (Seattle Genetics); SEA-CD40 (SeattleGenetics); ibritumomab tiuxetan (Spectrum); MLN0264 (Takeda); ganitumab(Takeda/Amgen); CEP-37250 (Teva); TB-403 (Thrombogenic); VB4-845(Viventia); Xmab2512 (Xencor); Xmab5574 (Xencor); nimotuzumab (YMBiosciences); Carlumab (Janssen); NY-ESO TCR (Adaptimmune); MAGE-A-10TCR (Adaptimmune); CTL019 (Novartis); JCAR015 (Juno Therapeutics);KTE-C19 CAR (Kite Pharma); UCART19 (Cellectis); BPX-401 (BellicumPharmaceuticals); BPX-601 (Bellicum Pharmaceuticals); ATTCK20 (UnumTherapeutics); CAR-NKG2D (Celyad); Onyx-015 (Onyx Pharmaceuticals); H101(Shanghai Sunwaybio); DNX-2401 (DNAtrix); VCN-01 (VCN Biosciences);Colo-Adl (PsiOxus Therapeutics); ProstAtak (Advantagene); Oncos-102(Oncos Therapeutics); CG0070 (Cold Genesys); Pexa-vac (JX-594, JennerexBiotherapeutics); GL-ONC1 (Genelux); T-VEC (Amgen); G207 (Medigene);HF10 (Takara Bio); SEPREHVIR (HSV1716, Virttu Biologics); OrienX010(OrienGene Biotechnology); Reolysin (Oncolytics Biotech); SVV-001(Neotropix); Cacatak (CVA21, Viralytics); Alimta (Eli Lilly), cisplatin,oxaliplatin, irinotecan, folinic acid, methotrexate, cyclophosphamide,5-fluorouracil, Zykadia (Novartis), Tafinlar (GSK), Xalkori (Pfizer),Iressa (AZ), Gilotrif (Boehringer Ingelheim), Tarceva (Astellas Pharma),Halaven (Eisai Pharma), Veliparib (Abbvie), AZD9291 (AZ), Alectinib(Chugai), LDK378 (Novartis), Genetespib (Synta Pharma),Tergenpumatucel-L (NewLink Genetics), GV1001 (Kael-GemVax), Tivantinib(ArQule); Cytoxan (BMS); Oncovin (Eli Lilly); Adriamycin (Pfizer);Gemzar (Eli Lilly); Xeloda (Roche); Ixempra (BMS); Abraxane (Celgene);Trelstar (Debiopharm); Taxotere (Sanofi); Nexavar (Bayer); IMMU-132(Immunomedics); E7449 (Eisai); Thermodox (Celsion); Cometriq (Exellxis);Lonsurf (Taiho Pharmaceuticals); Camptosar (Pfizer); UFT (TaihoPharmaceuticals); and TS-1 (Taiho Pharmaceuticals).

In some embodiments, the one or more bacterial strains having a 16s rRNAsequence that is at least 95% identical to SEQ ID NO:2, for examplewhich is an Enterococcus gallinarum, is/are the only therapeuticallyactive agent(s) in a composition of the invention. In some embodiments,the bacterial strain(s) in the composition is/are the onlytherapeutically active agent(s) in a composition of the invention.

Modes of Administration

Preferably, the compositions of the invention are to be administered tothe gastrointestinal tract in order to enable delivery to and/or partialor total colonization of the intestine with the bacterial strain of theinvention. Generally, the compositions of the invention are administeredorally, but they may be administered rectally, intranasally, or viabuccal or sublingual routes.

In certain embodiments, the compositions of the invention may beadministered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may beadministered as a suppository, such as a rectal suppository, for examplein the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g.suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soapglycerin composition.

In certain embodiments, the composition of the invention is administeredto the gastrointestinal tract via a tube, such as a nasogastric tube,orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneousendoscopic gastrostomy (PEG), or a port, such as a chest wall port thatprovides access to the stomach, jejunum and other suitable access ports.

The compositions of the invention may be administered once, or they maybe administered sequentially as part of a treatment regimen. In certainembodiments, the compositions of the invention are to be administereddaily.

In certain embodiments of the invention, treatment according to theinvention is accompanied by assessment of the subject's gut microbiota.Treatment may be repeated if delivery of and/or partial or totalcolonization with the strain of the invention is not achieved such thatefficacy is not observed, or treatment may be ceased if delivery and/orpartial or total colonization is successful and efficacy is observed.

In certain embodiments, the composition of the invention may beadministered to a pregnant animal, for example a mammal such as a humanin order to reduce the likelihood of cancer developing in her child inutero and/or after it is born.

The compositions of the invention may be administered to a subject thathas been diagnosed with cancer, or that has been identified as being atrisk of a cancer. The compositions may also be administered as aprophylactic measure to prevent the development of cancer in a healthysubject.

The compositions of the invention may be administered to a subject thathas been identified as having an abnormal gut microbiota. For example,the subject may have reduced or absent colonization by Enterococcusgallinarum.

The compositions of the invention may be administered as a food product,such as a nutritional supplement.

Generally, the compositions of the invention are for the treatment ofhumans, although they may be used to treat animals including monogastricmammals such as poultry, pigs, cats, dogs, horses or rabbits. Thecompositions of the invention may be useful for enhancing the growth andperformance of animals. If administered to animals, oral gavage may beused.

Compositions

Generally, the composition of the invention comprises bacteria. Inpreferred embodiments of the invention, the composition is formulated infreeze-dried form. For example, the composition of the invention maycomprise granules or gelatin capsules, for example hard gelatincapsules, comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilizedbacteria. Lyophilization of bacteria is a well-established procedure andrelevant guidance is available in, for example, references [26-28].

Alternatively, the composition of the invention may comprise a live,active bacterial culture.

In some embodiments, the bacterial strain in the composition of theinvention has not been inactivated, for example, has not beenheat-inactivated. In some embodiments, the bacterial strain in thecomposition of the invention has not been killed, for example, has notbeen heat-killed. In some embodiments, the bacterial strain in thecomposition of the invention has not been attenuated, for example, hasnot been heat-attenuated. For example, in some embodiments, thebacterial strain in the composition of the invention has not beenkilled, inactivated and/or attenuated. For example, in some embodiments,the bacterial strain in the composition of the invention is live. Forexample, in some embodiments, the bacterial strain in the composition ofthe invention is viable. For example, in some embodiments, the bacterialstrain in the composition of the invention is capable of partially ortotally colonizing the intestine. For example, in some embodiments, thebacterial strain in the composition of the invention is viable andcapable of partially or totally colonizing the intestine.

In some embodiments, the composition comprises a mixture of livebacterial strains and bacterial strains that have been killed.

In preferred embodiments, the composition of the invention isencapsulated to enable delivery of the bacterial strain to theintestine. Encapsulation protects the composition from degradation untildelivery at the target location through, for example, rupturing withchemical or physical stimuli such as pressure, enzymatic activity, orphysical disintegration, which may be triggered by changes in pH. Anyappropriate encapsulation method may be used. Exemplary encapsulationtechniques include entrapment within a porous matrix, attachment oradsorption on solid carrier surfaces, self-aggregation by flocculationor with cross-linking agents, and mechanical containment behind amicroporous membrane or a microcapsule. Guidance on encapsulation thatmay be useful for preparing compositions of the invention is availablein, for example, references [29] and [30].

The composition may be administered orally and may be in the form of atablet, capsule or powder. Encapsulated products are preferred becauseEnterococcus gallinarum are anaerobes. Other ingredients (such asvitamin C, for example), may be included as oxygen scavengers andprebiotic substrates to improve the delivery and/or partial or totalcolonization and survival in vivo. Alternatively, the probioticcomposition of the invention may be administered orally as a food ornutritional product, such as milk or whey based fermented dairy product,or as a pharmaceutical product.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effectiveamount of a bacterial strain of the invention. A therapeuticallyeffective amount of a bacterial strain is sufficient to exert abeneficial effect upon a subject. A therapeutically effective amount ofa bacterial strain may be sufficient to result in delivery to and/orpartial or total colonization of the subject's intestine.

A suitable daily dose of the bacteria, for example for an adult human,may be from about 1×10³ to about 1×10¹¹ colony forming units (CFU); forexample, from about 1×10⁷ to about 1×10¹⁰ CFU; in another example fromabout 1×10⁶ to about 1×10¹⁰ CFU.

In certain embodiments, the composition contains the bacterial strain inan amount of from about 1×10⁶ to about 1×10¹¹ CFU/g, respect to theweight of the composition; for example, from about 1×10⁸ to about 1×10¹⁰CFU/g. The dose may be, for example, 1 g, 3 g, 5 g, and 10 g of saidcomposition.

Typically, a probiotic, such as the composition of the invention, isoptionally combined with at least one suitable prebiotic compound. Aprebiotic compound is usually a non-digestible carbohydrate such as anoligo- or polysaccharide, or a sugar alcohol, which is not degraded orabsorbed in the upper digestive tract. Known prebiotics includecommercial products such as inulin and transgalacto-oligosaccharides.

In certain embodiments, the probiotic composition of the presentinvention includes a prebiotic compound in an amount of from about 1 toabout 30% by weight, respect to the total weight composition, (e.g. from5 to 20% by weight). Carbohydrates may be selected from the groupconsisting of: fructo-oligosaccharides (or FOS), short-chainfructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins,xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS),beta-glucans, arable gum modified and resistant starches, polydextrose,D-tagatose, acacia fibers, carob, oats, and citrus fibers. In oneaspect, the prebiotics are the short-chain fructo-oligosaccharides (forsimplicity shown herein below as FOSs-c.c); said FOSs-c.c. are notdigestible carbohydrates, generally obtained by the conversion of thebeet sugar and including a saccharose molecule to which three glucosemolecules are bonded.

The compositions of the invention may comprise pharmaceuticallyacceptable excipients or carriers. Examples of such suitable excipientsmay be found in the reference [31]. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art and aredescribed, for example, in reference [32]. Examples of suitable carriersinclude lactose, starch, glucose, methyl cellulose, magnesium stearate,mannitol, sorbitol and the like. Examples of suitable diluents includeethanol, glycerol and water. The choice of pharmaceutical carrier,excipient or diluent can be selected with regard to the intended routeof administration and standard pharmaceutical practice. Thepharmaceutical compositions may comprise as, or in addition to, thecarrier, excipient or diluent any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), solubilizing agent(s). Examplesof suitable binders include starch, gelatin, natural sugars such asglucose, anhydrous lactose, free-flow lactose, beta-lactose, cornsweeteners, natural and synthetic gums, such as acacia, tragacanth orsodium alginate, carboxymethyl cellulose and polyethylene glycol.Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Preservatives, stabilizers, dyes and even flavoring agents maybe provided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

The compositions of the invention may be formulated as a food product.For example, a food product may provide nutritional benefit in additionto the therapeutic effect of the invention, such as in a nutritionalsupplement. Similarly, a food product may be formulated to enhance thetaste of the composition of the invention or to make the compositionmore attractive to consume by being more similar to a common food item,rather than to a pharmaceutical composition. In certain embodiments, thecomposition of the invention is formulated with a nutritious productsuch as a milk-food product or a milk-based product. The term“milk-based product” means any liquid or semi-solid milk- or whey-basedproduct having a varying fat content. The milk-based product can be,e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, whole milk,milk recombined from powdered milk and whey without any processing, or aprocessed product, such as yogurt, curdled milk, curd, sour milk, sourwhole milk, butter milk and other sour milk products. Another importantgroup includes milk beverages, such as whey beverages, fermented milks,condensed milks, infant or baby milks; flavored milks, ice cream;milk-containing food such as sweets.

In certain embodiments, the compositions of the invention contain asingle bacterial strain or species and do not contain any otherbacterial strains or species. Such compositions may comprise only deminimis or biologically irrelevant amounts of other bacterial strains orspecies. Such compositions may be a culture that is substantially freefrom other species of organism. Thus, in some embodiments, the inventionprovides a composition comprising one or more strains from the speciesEnterococcus gallinarum, which does not contain bacteria from any otherspecies or which comprises only de minimis or biologically irrelevantamounts of bacteria from another species for use in therapy. In someembodiments, the composition consists essentially of Enterococcusgallinarum.

In some embodiments, the compositions of the invention comprise morethan one bacterial strain or species. For example, in some embodiments,the compositions of the invention comprise more than one strain fromwithin the same species (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40 or 45 strains), and, optionally, do not containbacteria from any other species. In some embodiments, the compositionsof the invention comprise less than 50 strains from within the samespecies (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6,5, 4 or 3 strains), and, optionally, do not contain bacteria from anyother species. In some embodiments, the compositions of the inventioncomprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6,1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15,16-25, or 31-50 strains from within the same species and, optionally, donot contain bacteria from any other species. In some embodiments, thecompositions of the invention comprise more than one species from withinthe same genus (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,17, 20, 23, 25, 30, 35 or 40 species), and, optionally, do not containbacteria from any other genus. In some embodiments, the compositions ofthe invention comprise less than 50 species from within the same genus(e.g. less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or3 species), and, optionally, do not contain bacteria from any othergenus. In some embodiments, the compositions of the invention comprise1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3,1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or31-50 species from within the same genus and, optionally, do not containbacteria from any other genus. The invention comprises any combinationof the foregoing.

In some embodiments, the composition comprises a microbial consortium.For example, in some embodiments, the composition comprises thebacterial strain having a 16s rRNA sequence that is at least 95%identical to SEQ ID NO:2, for example, which is an Enterococcusgallinarum, as part of a microbial consortium. For example, in someembodiments, the bacterial strain is present in combination with one ormore (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterial strainsfrom other genera with which it can live symbiotically in vivo in theintestine. For example, in some embodiments, the composition comprises abacterial strain having a 16s rRNA sequence that is at least 95%identical to SEQ ID NO:2, for example, which is an Enterococcusgallinarum, in combination with a bacterial strain from a differentgenus. In some embodiments, the microbial consortium comprises two ormore bacterial strains obtained from a faeces sample of a singleorganism, e.g. a human. In some embodiments, the microbial consortium isnot found together in nature. For example, in some embodiments, themicrobial consortium comprises bacterial strains obtained from faecessamples of at least two different organisms. In some embodiments, thetwo different organisms are from the same species, e.g. two differenthumans, e.g. two different human infants. In some embodiments, the twodifferent organisms are an infant human and an adult human. In someembodiments, the two different organisms are a human and a non-humanmammal.

In some embodiments, the composition of the invention additionallycomprises a bacterial strain that has the same safety and therapeuticefficacy characteristics as strain MRX518, but which is not MRX518deposited as NCIMB 42488, or which is not an Enterococcus gallinarum.

In some embodiments in which the composition of the invention comprisesmore than one bacterial strain, species or genus, the individualbacterial strains, species or genera may be for separate, simultaneousor sequential administration. For example, the composition may compriseall of the more than one bacterial strain, species or genera, or thebacterial strains, species or genera may be stored separately and beadministered separately, simultaneously or sequentially. In someembodiments, the more than one bacterial strains, species or genera arestored separately but are mixed together prior to use.

In some embodiments, the bacterial strain for use in the invention isobtained from human infant faeces. In some embodiments in which thecomposition of the invention comprises more than one bacterial strain,all of the bacterial strains are obtained from human infant faeces or ifother bacterial strains are present they are present only in de minimisamounts. The bacteria may have been cultured subsequent to beingobtained from the human infant faeces and being used in a composition ofthe invention.

The compositions for use in accordance with the invention may or may notrequire marketing approval.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilized. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is spray dried. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilized or spray driedand wherein it is live. In certain embodiments, the invention providesthe above pharmaceutical composition, wherein the bacterial strain islyophilized or spray dried and wherein it is viable. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilized or spray driedand wherein it is capable of partially or totally colonizing theintestine. In certain embodiments, the invention provides the abovepharmaceutical composition, wherein the bacterial strain is lyophilizedor spray dried and wherein it is viable and capable of partially ortotally colonizing the intestine.

In some cases, the lyophilized or spray dried bacterial strain isreconstituted prior to administration. In some cases, the reconstitutionis by use of a diluent described herein.

The compositions of the invention can comprise pharmaceuticallyacceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain as used in the invention; anda pharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder isbreast cancer. In preferred embodiments the cancer is mammary carcinoma.In preferred embodiments the cancer is stage IV breast cancer.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain as used in the invention; anda pharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder islung cancer. In preferred embodiments the cancer is lung carcinoma.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain as used in the invention; anda pharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder isliver cancer. In preferred embodiments the cancer is hepatoma(hepatocellular carcinoma).

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder iscolon cancer. In preferred embodiments the cancer is colorectaladenocarcinoma.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder iscarcinoma.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder is anon-immunogenic cancer.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder isselected from the group consisting of non-small-cell lung carcinoma,small-cell lung carcinoma, squamous-cell carcinoma, adenocarcinoma,glandular tumors, carcinoid tumors undifferentiated carcinomas.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder isselected from the group consisting of hepatoblastoma,cholangiocarcinoma, cholangiocellular cystadenocarcinoma or liver cancerresulting from a viral infection.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder isselected from the group consisting of invasive ductal carcinoma, ductalcarcinoma in situ or invasive lobular carcinoma.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder isselected from the group consisting of acute lymphoblastic leukemia(ALL), acute myeloid leukemia, adrenocortical carcinoma, basal-cellcarcinoma, bile duct cancer, bladder cancer, bone tumor,osteosarcoma/malignant fibrous histiocytoma, brainstem glioma, braintumor, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, breast cancer, bronchial adenomas/carcinoids, Burkitt'slymphoma, carcinoid tumor, cervical cancer, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic myeloproliferativedisorders, colon cancer, cutaneous T-cell lymphoma, endometrial cancer,ependymoma, esophageal cancer, Ewing's sarcoma, intraocular melanoma,retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinalcarcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor,glioma, childhood visual pathway and hypothalamic, Hodgkin lymphoma,melanoma, islet cell carcinoma, Kaposi sarcoma, renal cell cancer,laryngeal cancer, leukaemias, lymphomas, mesothelioma, neuroblastoma,non-Hodgkin lymphoma, oropharyngeal cancer, osteosarcoma, ovariancancer, pancreatic cancer, parathyroid cancer, pharyngeal cancer,pituitary adenoma, plasma cell neoplasia, prostate cancer, renal cellcarcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer,or uterine cancer.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the amount of the bacterial strain is from about1×10³ to about 1×10¹¹ colony forming units per gram with respect to aweight of the composition.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered at a dose of 1 g, 3g, 5 g or 10 g.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered by a methodselected from the group consisting of oral, rectal, subcutaneous, nasal,buccal, and sublingual.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a carrier selected from the group consisting oflactose, starch, glucose, methyl cellulose, magnesium stearate, mannitoland sorbitol.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a diluent selected from the group consisting ofethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising an excipient selected from the group consistingof starch, gelatin, glucose, anhydrous lactose, free-flow lactose,beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate,carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate and sodiumchloride.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, further comprising at least one of a preservative, anantioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a preservative selected from the groupconsisting of sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein when the composition is stored in a sealedcontainer at about 4.0 or about 25.0 and the container is placed in anatmosphere having 50% relative humidity, at least 80% of the bacterialstrain as measured in colony forming units, remains after a period of atleast about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years,2.5 years or 3 years.

In some embodiments, the composition of the invention is provided in asealed container comprising a composition as described herein. In someembodiments, the sealed container is a sachet or bottle. In someembodiments, the composition of the invention is provided in a syringecomprising a composition as described herein.

The composition of the present invention may, in some embodiments, beprovided as a pharmaceutical formulation. For example, the compositionmay be provided as a tablet or capsule. In some embodiments, the capsuleis a gelatine capsule (“gel-cap”).

In some embodiments, the compositions of the invention are administeredorally. Oral administration may involve swallowing, so that the compoundenters the gastrointestinal tract, and/or buccal, lingual, or sublingualadministration by which the compound enters the blood stream directlyfrom the mouth.

Pharmaceutical formulations suitable for oral administration includesolid plugs, solid microparticulates, semi-solid and liquid (includingmultiple phases or dispersed systems) such as tablets; soft or hardcapsules containing multi- or nano-particulates, liquids (e.g. aqueoussolutions), emulsions or powders; lozenges (including liquid-filled);chews; gels; fast dispersing dosage forms; films; ovules; sprays; andbuccal/mucoadhesive patches.

In some embodiments the pharmaceutical formulation is an entericformulation, i.e. a gastro-resistant formulation (for example, resistantto gastric pH) that is suitable for delivery of the composition of theinvention to the intestine by oral administration. Enteric formulationsmay be particularly useful when the bacteria or another component of thecomposition is acid-sensitive, e.g. prone to degradation under gastricconditions.

In some embodiments, the enteric formulation comprises an entericcoating. In some embodiments, the formulation is an enteric-coateddosage form. For example, the formulation may be an enteric-coatedtablet or an enteric-coated capsule, or the like. The enteric coatingmay be a conventional enteric coating, for example, a conventionalcoating for a tablet, capsule, or the like for oral delivery. Theformulation may comprise a film coating, for example, a thin film layerof an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the enteric formulation is intrinsically enteric,for example, gastro-resistant without the need for an enteric coating.Thus, in some embodiments, the formulation is an enteric formulationthat does not comprise an enteric coating. In some embodiments, theformulation is a capsule made from a thermogelling material. In someembodiments, the thermogelling material is a cellulosic material, suchas methylcellulose, hydroxymethylcellulose orhydroxypropylmethylcellulose (HPMC). In some embodiments, the capsulecomprises a shell that does not contain any film forming polymer. Insome embodiments, the capsule comprises a shell and the shell compriseshydroxypropylmethylcellulose and does not comprise any film formingpolymer (e.g. see [33]). In some embodiments, the formulation is anintrinsically enteric capsule (for example, Vcaps® from Capsugel).

In some embodiments, the formulation is a soft capsule. Soft capsulesare capsules which may, owing to additions of softeners, such as, forexample, glycerol, sorbitol, maltitol and polyethylene glycols, presentin the capsule shell, have a certain elasticity and softness. Softcapsules can be produced, for example, on the basis of gelatine orstarch. Gelatine-based soft capsules are commercially available fromvarious suppliers. Depending on the method of administration, such as,for example, orally or rectally, soft capsules can have various shapes,they can be, for example, round, oval, oblong or torpedo-shaped. Softcapsules can be produced by conventional processes, such as, forexample, by the Scherer process, the Accogel process or the droplet orblowing process.

Culturing Methods

The bacterial strains for use in the present invention can be culturedusing standard microbiology techniques as detailed in, for example,references [34-36].

The solid or liquid medium used for culture may be YCFA agar or YCFAmedium. YCFA medium may include (per 100 ml, approximate values):Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g), cysteine (0.1g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g), (NH₄)₂SO₄ (0.09g), MgSO₄.7H₂O (0.009 g), CaCl₂ (0.009 g), resazurin (0.1 mg), hemin (1mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoic acid (3 μg), folicacid (5 μg), and pyridoxamine (15 μg).

Bacterial Strains for Use in Vaccine Compositions

The inventors have identified that the bacterial strains of theinvention are useful for treating or preventing cancer. This is likelyto be a result of the effect that the bacterial strains of the inventionhave on the host immune system. Therefore, the compositions of theinvention may also be useful for preventing cancer, when administered asvaccine compositions. In certain such embodiments, the bacterial strainsof the invention are viable. In certain such embodiments, the bacterialstrains of the invention are capable of partially or totally colonizingthe intestine. In certain such embodiments, the bacterial strains of theinvention are viable and capable of partially or totally colonizing theintestine. In other certain such embodiments, the bacterial strains ofthe invention may be killed, inactivated or attenuated. In certain suchembodiments, the compositions may comprise a vaccine adjuvant. Incertain embodiments, the compositions are for administration viainjection, such as via subcutaneous injection.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references[37] and [38-44], etc.

A subject treated by a method described herein, or by contact with oradministration of a composition described herein can be a mammaliansubject who can be a human subject, a non-human primate, a caninemammal, a felid mammal or any other mammal. A subject maybe a patientwho is a mammalian patient for instance, a human patient, a non-humanprimate, a canine mammal, a felid mammal or any other mammalian patient.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to a percentage sequence identity between two nucleotidesequences means that, when aligned, that percentage of nucleotides arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref[45]. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref [46].

Unless specifically stated, a process or method comprising numeroussteps may comprise additional steps at the beginning or end of themethod, or may comprise additional intervening steps. Also, steps may becombined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will beappreciated that the features specified in each embodiment may becombined with other specified features, to provide further embodiments.In particular, embodiments highlighted herein as being suitable, typicalor preferred may be combined with each other (except when they aremutually exclusive).

MODES FOR CARRYING OUT THE INVENTION Example 1—Efficacy of BacterialInocula in Mouse Models of Cancer

Summary

This study tested the efficacy of compositions comprising an exemplarybacterial strain according to the invention in four tumor models.

Materials

Test Substance—

Bacterial strain #MRX518.

Reference Substance—

Anti-CTLA-4 antibody (clone: 9H10, catalog: BE0131, isotype: SyrianHamster IgG1, Bioxcell).

Test and Reference Substances Vehicles—

Bacterial culture medium (Yeast extract, Casitone, Fatty Acid medium(YCFA)). Each day of injection to mice, antibody was diluted with PBS(ref: BE14-516F, Lonza, France).

Treatment Doses—

Bacteria: 2×10⁸ in 200 μL. The a-CTLA-4 was injected at 10 mg/kg/inj.Anti-CTLA-4 was administered at a dose volume of 10 mL/kg/adm (i.e. forone mouse weighing 20 g, 200 μL of test substance will be administered)according to the most recent body weight of mice.

Routes of Administration—

Bacterial inoculum was administered by oral gavage (per os, PO) via acannula. Cannulas were decontaminated every day. Anti-CTLA-4 wasinjected into the peritoneal cavity of mice (Intraperitoneally, IP).

Culture Conditions of Bacterial Strain—

The culture conditions for the bacterial strain were as follows:

-   -   Pipette 10 mL of YCFA (from the prepared 10 mL E&O lab bottles)        into Hungate tubes    -   Seal the tubes and flush with CO₂ using a syringe input and        exhaust system    -   Autoclave the Hungate tubes    -   When cooled, inoculate the Hungate tubes with 1 mL of the        glycerol stocks    -   Place the tubes in a static 37° C. incubator for about 16 hours.    -   The following day, take 1 mL of this subculture and inoculate 10        mL of YCFA (pre-warmed flushed Hungate tubes again, all in        duplicate)    -   Place them in a static 37° C. incubator for 5 to 6 h

Cancer Cell Line and Culture Conditions—

The cell lines that were used are detailed in the table below:

Cell Mouse line Type strain Origin EMT-6 Breast BALB/c ATCC carcinomaLL/2 (LLC1) Lung C57BL/6 ATCC CRL1642 carcinoma Hepal-6 HepatocellularC57BL/6 IPSEN INNOVATION carcinoma

The EMT-6 cell line was established from a transplantable murine mammarycarcinoma that arose in a BALB/cCRGL mouse after implantation of ahyperplastic mammary alveolar nodule [47].

The LL/2 (LLC1) cell line was established from the lung of a C57BL mousebearing a tumor resulting from an implantation of primary Lewis lungcarcinoma [48].

The Hepa 1-6 cell line is a derivative of the BW7756 mouse hepatoma thatarose in a C57/L mouse [49].

Cell Culture Conditions—

All cell lines were grown as monolayer at 37° C. in a humidifiedatmosphere (5% CO₂, 95% air). The culture medium and supplement areindicated in the table below:

Cell line Culture medium Supplement EMT6 RPMI 1640 containing 2 mM 10%fetal bovine serum L-glutamine (ref: #3302, Lonza) (ref: BE12-702F,Lonza) LL/2 RPMI 1640 containing 2 mM 10% fetal bovine serum (LLC1)L-glutamine (ref: #3302, Lonza) (ref: BE12-702F, Lonza) Hepal-6 DMEM 10%fetal bovine serum (ref:11960-044, Gibco) (ref: #3302, Lonza) 2 mML-Glutamine penicillin-streptomycin (Sigma G-6784)

For experimental use, adherent tumor cells were detached from theculture flask by a 5 minute treatment with trypsin-versene (ref:BE17-161E, Lonza), in Hanks' medium without calcium or magnesium (ref:BE10-543F, Lonza) and neutralized by addition of complete culturemedium. The cells were counted in a hemocytometer and their viabilitywill be assessed by 0.25% trypan blue exclusion assay.

Use of Animals—

Healthy female Balb/C (BALB/cByJ) mice, of matching weight and age, wereobtained from CHARLES RIVER (L'Arbresles) for the EMT6 modelexperiments.

Healthy female C57BL/6 (C57BL16J) mice, of matching weight and age, wereobtained from CHARLES RIVER (L'Arbresles) for the LL/2(LLC1) and theHepa1-6 model experiments.

Animals were maintained in SPF health status according to the FELASAguidelines, and animal housing and experimental procedures according tothe French and European Regulations and NRC Guide for the Care and Useof Laboratory Animals were followed [50,51]. Animals were maintained inhousing rooms under controlled environmental conditions: Temperature:22±2° C., Humidity 55±10%, Photoperiod (12 h light/12 h dark), HEPAfiltered air, 15 air exchanges per hour with no recirculation. Animalenclosures were provided with sterile and adequate space with beddingmaterial, food and water, environmental and social enrichment (grouphousing) as described: 900 cm² cages (ref: green, Tecniplast) inventilated racks, Epicea bedding (SAFE), 10 kGy Irradiated diet (A04-10,SAFE), Complete food for immuno-competent rodents—R/M-H Extrudate, waterfrom water bottles.

Experimental Design and Treatments

Antitumor Activity, EMT6 Model

Treatment Schedule—

The start of first dosing was considered as D0. On D0, non-engraftedmice were randomized according to their individual body weight intogroups of 9/8 using Vivo Manager® software (Biosystemes, Couternon,France). On D0, the mice received vehicle (culture medium) or bacterialstrain. On D14, all mice were engrafted with EMT-6 tumor cells asdescribed below. On D24, mice from the positive control group receivedanti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

No. Treatment Group Animals Treatment Dose Route Schedule 1 8 Untreated— — — 2 8 Vehicle (media) — PO Q1Dx42 3 9 Bacterial strain #1 2 × 10⁸ POQ1Dx42 (MRX518) bacteria 4 8 Anti-CTLA4 10 mg/kg IP TWx2

The monitoring of animals was performed as described below.

Induction of EMT6 Tumors in Animals—

On D14, tumors were induced by subcutaneous injection of 1×10⁶ EMT-6cells in 200 μL RPMI 1640 into the right flank of mice.

Euthanasia—

Each mouse was euthanized when it reached a humane endpoint as describedbelow, or after a maximum of 6 weeks post start of dosing.

Antitumor Activity, LL/2 (LLC1) Model

Treatment Schedule—

The start of first dosing was considered as D0. On D0, non-engraftedmice were randomized according to their individual body weight into 7groups of 9/8 using Vivo Manager® software (Biosystemes, Couternon,France). On D0, the mice will received vehicle (culture medium) orbacterial strain. On D14, all mice were engrafted with LL/2 tumor cellsas described below. On D27, mice from the positive control groupreceived anti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

No. Treatment Group Animals Treatment Dose Route Schedule 1 8 Untreated— — — 2 9 Vehicle (media) — PO Q1Dx42 3 9 Bacterial strain #1 2 × 10⁸ POQ1Dx42 (MRX518) bacteria 4 8 Anti-CTLA4 10 mg/kg IP TWx2

The monitoring of animals was performed as described below.

Induction of LL/2 (LLC1) Tumors in Animals—

On D14, tumors were induced by subcutaneous injection of 1×10⁶ LL/2(LLC1) cells in 200 μL RPMI 1640 into the right flank of mice.

Euthanasia—

Each mouse was euthanized when it reached a humane endpoint as describedbelow, or after a maximum of 6 weeks post start of dosing.

Antitumor Activity, Hepa1-6 Model

Treatment Schedule—

The start of first dosing was considered as D0. On D0, non-engraftedmice were randomized according to their individual body weight into 7groups of 9 using Vivo Manager® software (Biosystemes, Couternon,France). On D0, the mice received vehicle (culture medium) or bacterialstrain. On D14, all mice were engrafted with Hepa 1-6 tumor cells asdescribed below. On D16, mice from the positive control group receivedanti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

No. Treatment Group Animals Treatment Dose Route Schedule 1 9 Untreated— — — 2 9 Vehicle (media) — PO Q1Dx42 6 9 Bacterial strain #4 2 × 10⁸ POQ1Dx42 (MRX518) bacteria 7 9 Anti-CTLA4 10 mg/kg IP TWx2

The monitoring of animals was performed as described below.

Orthotopic Induction of Hepa 1-6 Tumor Cells in Animals by IntrasplenicInjection—

On D14, one million (1×10⁶) Hepa 1-6 tumor cells in 50 μL RPMI 1640medium were transplanted via intra-splenic injection into mice. Briefly,a small left subcostal flank incision was made and the spleen wasexteriorized. The spleen was exposed on a sterile gauze pad, andinjected under visual control with the cell suspension with a 27-gaugeneedle. After the cell inoculation, the spleen was excised.

Euthanasia—

Each mouse was euthanized when it reached a humane endpoint as describedin section below, or after a maximum of 6 weeks post start of dosing.

Evaluation of Tumor Burden at Euthanasia—

At the time of termination, livers were collected and weighed.

Animal Monitoring

Clinical Monitoring—

The length and width of the tumor was measured twice a week withcallipers and the volume of the tumor was estimated by this formula[52]:

${Tumor}\mspace{14mu}{volume}{= \frac{{width}^{2} \times {length}}{2}}$

Humane Endpoints [53]:

Signs of pain, suffering or distress: pain posture, pain face mask,behaviour; Tumor exceeding 10% of normal body weight, but non-exceeding2000 mm³; Tumors interfering with ambulation or nutrition; Ulceratedtumor or tissue erosion; 20% body weight loss remaining for 3consecutive days; Poor body condition, emaciation, cachexia,dehydration; Prolonged absence of voluntary responses to externalstimuli; Rapid laboured breathing, anaemia, significant bleeding;Neurologic signs: circling, convulsion, paralysis; Sustained decrease inbody temperature; Abdominal distension.

Anaesthesia—

Isoflurane gas anesthesia were used for all procedures: surgery or tumorinoculation, i.v. injections, blood collection. Ketamine and Xylazineanesthesia were used for stereotaxia surgical procedure.

Analgesia—

Carprofen or multimodal carprofen/buprenorphine analgesia protocol wereadapted to the severity of surgical procedure. Non-pharmacological carewas provided for all painful procedures. Additionally, pharmacologicalcare not interfering with studies (topic treatment) were provided at therecommendation of the attending veterinarian.

Euthanasia—

Euthanasia of animals was performed by gas anesthesia over-dosage(Isoflurane) followed by cervical dislocation or exsanguination.

Results

Antitumor Activity, EMT6 Model

The results are shown in FIG. 1. Treatment with an exemplary bacterialstrain of the invention led to a clear reduction in tumor volumerelative to both the negative controls. The positive control also led toa reduction in tumor volume, as would be expected.

Antitumor Activity, LL/2 (LLC1) Model

The results are shown in FIG. 2. Treatment with an exemplary bacterialstrain of the invention led to a clear reduction in tumor volumerelative to both the negative controls.

Antitumor Activity, Hepa1-6 Model

The results are shown in FIG. 3. The untreated negative control does notappear as would be expected, because liver weight was lower in thisgroup than the other groups. However, the vehicle negative control andthe positive control groups both appear as would be expected, becausemice treated with vehicle alone had larger livers than mice treated withanti-CTLA4 antibodies, reflecting a greater tumor burden in the vehiclenegative control group. Treatment with an exemplary bacterial strain ofthe invention led to a clear reduction in liver weight (and thereforetumor burden) relative to the mice in the vehicle negative controlgroup.

These data indicate that strain MRX518 may be useful for treating orpreventing cancer, and in particular for reducing tumor volume inbreast, lung and liver cancers.

Example 2—PCR Gene Analysis

A pure culture of bacteria MRX518 was studied in a PCR gene analysis.There were two arms to the experiment: 1) MRX518 was co-cultured withhuman colonic cells (CaCo2) to investigate the effects of the bacteriaon the host, and 2) MRX518 was co-cultured on CaCo2 cells that werestimulated with IL1 to mimic the effect of the bacteria in aninflammatory environment. The effects in both scenarios were evaluatedthrough gene expression analysis. The results are shown below:

Fold Gene change Function CXCL3 28412.73 CXCR2 ligand, CXCL2 135.42CXCR2 ligand, 90% homology with CXCL1. CXCL9 34.76 CXCR3 ligand,primarily thought of as Th1 cell chemoattractant (inducible by IFN-g)IL8 31.81 Cytokine, chemoattractant (especially neutrophils), manyreceptors including CXCR1 and CXCR2/ CXCL1 16.48 CXCR2 ligand,stimulates cell proliferation as well as migration, overexpression isneuroprotective in EAE. CD40 14.33 Co-stimulatory molecule, route of Tcell dependent DC activation. TNF 13.50 Major proinflammatory cytokineIL17C 12.18 Promotes antibacterial response from epthielium, synergisticwith IL-22, CXCL10 10.66 Close homology with CXCL9, think also CXCR3ligand HSPA1B 10.19 Heat shock protein NFKBIA 8.87 NFkB signalling; PI3KJUN 7.61 Antibacterial response; GPCR signalling. TNFAIP3 6.63 TNFsignalling DUSP1 6.36 Anti-inflammatory phosphatase, inactivates MAPKsJUNB 5.36 Transcription factor, JAK-STAT signalling BIRC3 4.86 Adherensjunctions, tight junctions DUSP2 4.59 Anti-inflammatory, inactivatesMAPK. IL32 4.29 Proinflammatory cytokine, induced by IFN-g, IL-18 DUSP53.12 Anti-inflammatory, inactivates MAPK FOS 3.03 Transcription factors,TLR signalling, forms part of AP-1 GADD45B 2.89 Cell growth andproliferation CLDN4 2.61 Tight junctions ADM 2.57 NFkB signalling KLF102.49 Cell arrest, TGF-b singllaing. DEFB4A −2.34 Antimicrobial peptideAPBA1 −2.53 Signalling IGFBP1 −2.72 Signalling pathway IL28B −2.73IFN-lambda, antiviral immune defence, IL10 −3.38 Anti-inflammatorycytokine NR4A1 −5.57 Nuclear receptor, anti-inflammatory, regulator of Tcell proliferation. T helper cell differentiation NOD2 −14.98 PRR,inflammasome activator, promotes autophagy INOS −26.88 Proinflammatory,generator of nitric oxide

These data appear to show two gene expression signatures—CXCR1/2 ligands(CXCL3, CXCL2, CXCL1, IL-8), which is associated with pro-inflammatorycell migration, and CXCR3 ligands (CXCL9, CXCL10), which is morespecifically indicative of IFN-γ-type responses, also supported byIL-32, which is IFN-γ-inducible.

Example 3—Stability Testing

A composition described herein containing at least one bacterial straindescribed herein is stored in a sealed container at 25° C. or 4° C. andthe container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%,75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, atleast 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain asmeasured in colony forming units determined by standard protocols.

Example 4—Cytokine Production in Immature Dendritic Cells Induced byMRX518 Compared to MRX518+LPS

Summary

This study tested the effect of the bacterial strain MRX518 alone and incombination with lipopolysaccharide (LPS) on cytokine production inimmature dendritic cells.

A monocyte population was isolated from peripheral blood mononuclearcells (PBMCs). The monocyte cells were subsequently differentiated intoimmature dendritic cells. The immature dendritic cells were plated outat 200,000 cells/well and incubated with MRX518 at a final concentrationof 10⁷/ml, with the optional addition of LPS at a final concentration of100 ng/ml. The negative control involved incubating the cells with RPMImedia alone and positive controls incubated the cells with LPS at afinal concentration of 100 ng/ml. The cytokine content of the cells wasthen analyzed.

Results

The results of these experiments can be seen in FIGS. 4a-d . Theaddition of MRX518 alone leads to a substantial increase in the level ofcytokines IL-6 and TNF-α compared to the negative control (FIGS. 4a andc ). The addition of LPS (positive control) leads to an increase in thelevel of IL-6 and TNF-α compared to the negative control but not IL-1β(FIG. 4b ). A combination of MRX518 and LPS led to a synergisticincrease in the level of IL-1β produced (FIG. 4d ).

Conclusion

MRX518 has the ability to induce higher IL-6 and TNF-α cytokineproduction in immature dendritic cells. The combination LPS and MRX518can increase the levels of cytokines IL-1β in immature dendritic cells.These data indicate that MRX518 alone or in combination with LPS canincrease inflammatory cytokines IL-1β, IL-6 and TNF-α, which promotesinflammation that can suppress cancer. Treatment with MRX518 alone or incombination with can induce cytokines that can limit tumor growth.

Example 5—Cytokine Production in THP-1 Cells Induced by MRX518 Comparedto MRX518+LPS

Summary

This study tested the effect of bacterial strain MRX518 alone and incombination with LPS on cytokine production in THP-1 cells, a model cellline for monocytes and macrophages.

THF-1 cells were differentiated into M0 medium for 48 h with 5 ng/mLphorbol-12-myristate-13-acetate (PMA). These cells were subsequentlyincubated with MRX518 at a final concentration of 10⁸/ml, with orwithout the addition of LPS at a final concentration of 100 ng/ml. Thebacteria were then washed off and the cells allowed to incubate undernormal growing conditions for 24 h. The cells were then spun down andthe resulting supernatant was analyzed for cytokine content.

Results

The results of these experiments can be seen in FIGS. 5a-c . Theaddition of MRX518 without LPS leads to an increase in the cytokinelevels of IL-1β, IL-6 and TNF-α compared to the no bacterial and thebacterial sediment controls. The addition of LPS and MRX518 leads to asynergistic increase in the production of cytokines.

Conclusion

MRX518 has the ability to induce cytokine production in THP-1 cells,which can be synergistically increased with the addition of LPS. Thesedata indicate that MRX518 alone or in combination with LPS can increaseinflammatory cytokines IL-1β, IL-6 and TNF-α, which promotesinflammation that can suppress cancer. Treatment with MRX518 alone or incombination with can induce cytokines that can limit tumor growth.

SEQUENCES SEQ ID NO: 1 (Enterococcus gallinarum 16S rRNA gene-AF039900)   1 taatacatgc aagtcgaacg ctttttcttt caccggagct tgctccaccg aaagaaaaag  61 agtggcgaac gggtgagtaa cacgtgggta acctgcccat cagaagggga taacacttgg 121 aaacaggtgc taataccgta taacactatt ttccgcatgg aagaaagttg aaaggcgctt 181 ttgcgtcact gatggatgga cccgcggtgc attagctagt tggtgaggta acggctcacc 241 aaggccacga tgcatagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 301 cccagactcc tacgggaggc agcagtaggg aatcttcggc aatggacgaa agtctgaccg 361 agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgt tagagaagaa 421 caaggatgag agtagaacgt tcatcccttg acggtatcta accagaaagc cacggctaac 481 tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggatt tattgggcgt 541 aaagcgagcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 601 gtcattggaa actgggagac ttgagtgcag aagaggagag tggaattcca tgtgtagcgg 661 tgaaatgcgt agatatatgg aggaacacca gtggcgaagg cggctctctg gtctgtaact 721 gacgctgagg ctcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 781 gtaaacgatg agtgctaagt gttggagggt ttccgccctt cagtgctgca gcaaacgcat 841 taagcactcc gcctggggag tacgaccgca aggttgaaac tcaaaggaat tgacgggggc 901 ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 961 ttgacatcct ttgaccactc tagagataga gcttcccctt cgggggcaaa gtgacaggtg1021 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca1081 acccttattg ttagttgcca tcatttagtt gggcactcta gcgagactgc cggtgacaaa1141 ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg1201 tgctacaatg ggaagtacaa cgagttgcga agtcgcgagg ctaagctaat ctcttaaagc1261 ttctctcagt tcggattgta ggctgcaact cgcctacatg aagccggaat cgctagtaat1321 cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac1381 cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt tttggagcca gccgcctaag1441 gtgggataga tgattggggt gaagtcgtaa caaggtagcc gtatcggaag gtgcggctgg1501 atcaccSEQ ID NO: 2 (consensus 16S rRNA sequence for Enterococcus gallinarumstrain MRX518) TGCTATACATGCAGTCGAACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAAAAAGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACACTATTTTCCGCATGGAAGAAAGTTGAAAGGCGCTTTTGCGTCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGCrCCACGATGCATAGCCGACCTGAGAGGGTGATCGCrCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAGAACGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTTGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGSEQ ID NO:3 (strain MRX518 chromosome sequence)—see electronic sequencelisting.SEQ ID NO:4 (strain MRX518 plasmid sequence)—see electronic sequencelisting.

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What is claimed is:
 1. A method of increasing a level of an IL-6 orTNF-α cytokine in a subject in need thereof, comprising administering tosaid subject a pharmaceutical composition that comprises: (a) a bacteriastrain of the species Enterococcus gallinarum that comprises a 16s rRNAgene sequence with at least 99.5% sequence identity to thepolynucleotide of SEQ ID NO: 2, wherein said sequence identity isdetermined by the Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, and a BLOSUM matrix of 62, and (b) a prebiotic compound,wherein said administering is sufficient to increase said level of saidIL-6 or TNF-α cytokine at least 100-fold relative to prior to saidadministering.
 2. The method of claim 1, wherein said administering issufficient to increase said level of said IL-6 cytokine.
 3. The methodof claim 2, wherein said administering is sufficient to increase saidlevel of said IL-6 cytokine at least 600-fold, relative to prior to saidadministering.
 4. The method of claim 1, wherein said administering issufficient to increase said level of said TNF-α cytokine.
 5. The methodof claim 4, wherein said administering is sufficient to increase saidlevel of said IL-6 cytokine at least 400-fold, relative to prior to saidadministering.
 6. The method of claim 1, wherein said pharmaceuticalcomposition further comprises a lipopolysaccharide.
 7. The method ofclaim 6, wherein said administering is sufficient to increase a level ofan IL-1β cytokine at least 1000-fold, relative to prior to saidadministering.
 8. The method of claim 1, wherein said amount of saidbacteria strain comprises from about 1×10³ to about 1×10¹¹ CFU/g of saidbacteria strain with respect to a total weight of said pharmaceuticalcomposition.
 9. The method of claim 1, wherein said pharmaceuticalcomposition is formulated for oral delivery.
 10. The method of claim 1,wherein said pharmaceutical composition is formulated for delivery to anintestine of said subject.
 11. The method of claim 1, wherein saidsubject is human.
 12. The method of claim 1, wherein said prebioticcompound is selected from the group consisting of: afructo-oligosaccharide, a short-chain fructo-oligosaccharide, inulin, anisomalt-oligosaccharide, a transgalacto-oligosaccharide, a pectin, axylo-oligosaccharide, a chitosan-oligosaccharide, a beta-glucan, anarable gum modified starch, a polydextrose, a D-tagatose, an acaciafiber, carob, an oat, and a citrus fiber.